INHIBITION BY ANTIOXIDANTS OF NITRIC-OXIDE SYNTHASE EXPRESSION IN MURINE MACROPHAGES - ROLE OF NUCLEAR FACTOR KAPPA-B AND INTERFERON REGULATORY FACTOR-1

1 In view of the potential deleterious effects of high amounts of nitr ic oxide (NO) produced by the inducible isoform of NO synthase (iNOS) in inflammation, the prevention of the expression of this enzyme repre sents an important therapeutic goal. In cytokine-stimulated cells, act ivation of nuclear factor kappa B (NF-kappa B) is crucial for the incr ease in iNOS gene expression. Since NF-kappa B activation appears to i nvolve a redox-sensitive step, we have investigated whether three stru cturally unrelated antioxidants, 5,7-dihydroxyflavone (chrysin), 3,4-d ichloroisocoumarin (DCI) and N-acetyl 5-hydroxytryptamine (N-acetylser otonin, NAS), affect iNOS expression in cultured RAW 264.7 monocyte/ma crophages stimulated with bacterial lipopolysaccharide (LPS, 140 ng ml (-1)) and interferon-gamma (IFN gamma, 5 u ml(-1)). 2 During a 6 h inc ubation period neither LPS nor IFN gamma alone exerted a significant e ffect but when combined, caused a prominent increase in nitrite format ion, iNOS mRNA and protein abundance. Coincubation with chrysin (50 mu M), DCI (50 mu M) or NAS (1 mM) markedly attenuated this increase in iNOS gene expression. 3 DCI, but not chrysin or NAS, prevented the act ivation of NF-kappa B in cells exposed to LPS plus IFN gamma, for 30 m in. In contrast, all three antioxidants significantly blunted the DNA- binding activity of interferon regulatory factor 1 (IRF-1), which medi ates the synergistic effect of IFN gamma on iNOS gene expression in ce lls treated for 2 h with LPS plus IFN gamma. 4 DCI thus appears to inh ibit iNOS gene expression at the transcriptional level by preventing t he activation of both NF-kappa B and IRF-1. The inhibitory effect of D CI on NF-kappa B activation, however, does not seem to be related to i ts antioxidative properties, since DCI, unlike chrysin or NAS, is a po tent serine protease inihbitor which stabilizes the inactive NF-kappa B complex by protecting the inhibitory I kappa B-alpha subunit from pr oteolytic degradation. 5 The virtually identical inhibitory effect of chrysin, DCI and NAS on the activation of IRF-1 points to a redox-sens itive step in the activation of this transcription factor, which in co ntrast to NF-kappa B requires de novo protein synthesis. 6 Since iNOS gene expression in human cells and tissues usually requires the combin ation of several cytokines, antioxidants such as chrysin and NAS which do not interfere with the activation of NF-kappa B may be of therapeu tic value for selectively inhibiting the enhanced expression of this e nzyme in inflammation.